CorrectionsBIOCHEMISTRY. For the article ''Interaction of RNA polymerase with forked DNA: Evidence for two kinetically significant intermediates on the pathway to the final complex,'' by Laura Tsujikawa, Oleg V. Tsodikov, and Pieter L. deHaseth, which appeared in number 6, March 19, 2002, of Proc. Natl. Acad. Sci. USA (99, 3493-3498; First Published March 12, 2002; 10.1073͞ pnas.062487299), the authors note the following concerning RNA polymerase (RNAP) concentrations. No correction was made for the fraction of RNAP (0.5) that is active in promoter binding. With this correction, the values of K 1 and K app (but not K f ) would increase by about a factor of 2. The relative values would remain essentially unchanged. Also, the legends to Figs. 2, 3, and 5 contain errors pertaining to the symbols used for data obtained with and without heparin challenge, the duration of the challenge, and the concentration of added heparin. The figures and the corrected legends appear below. Fig. 2. Determination of equilibrium affinities by titration of wt Fork with RNAP. The reactions contained 1 nM wt Fork and variable amounts of RNAP as shown and were analyzed by electrophoretic mobility shift immediately (OE; data shown are averages of three independent experiments) or after a challenge with 100 g͞ml heparin for 10 min (F; data shown are averages of four independent experiments). The curves shown reflect the simultaneous errorweighted fits of the data to Eqs. 3 and 4 -7. The parameters are shown in Table 1 (line 1). www.pnas.org͞cgi͞doi͞10.1073͞pnas.013667699 Fig. 3. Kinetics of complex formation. RNAP (65 nM) and wt forked DNA (1 nM) were incubated for various time intervals and then complex formation was determined immediately (Ϫheparin) or after a 2-min challenge with 100 g͞ml heparin (ϩheparin). The Ϫheparin data (s) were fit (error-weighted) with Eq. 8 with a 2 ϭ 0 (kaϪ ϭ 0.10 Ϯ 0.01 s Ϫ1 ) and the ϩheparin data (OE) with both single (k aϩ ϭ 0.036 Ϯ 0.004 s Ϫ1 ; thin line) and double-exponential (ka 1 ϭ 0.044 Ϯ 0.002 s Ϫ1 ; ka 2 ϭ (5 Ϯ 3) ϫ 10 Ϫ4 s Ϫ1 ; thick line) equations. Fig. 5.Comparison of the kinetics for formation and dissociation of competitor-resistant complexes between RNAP and wt Fork. Association data were obtained as described in the text and the legend for Fig. 3 except the concentration of forked DNA was 10 nM. Dissociation kinetics were obtained by challenging with 100 g͞ml heparin a mixture of RNAP and forked DNA that had been incubated for 30 min. The curves represent double-exponential fits of the data to Eq. 10. (A) wt RNAP. The observed association rate constants (s) are shown in the legend for Fig. 3; for the slow phase of the dissociation of the wt Fork-wt RNAP complex (F), kd 2 ϭ (1.3 Ϯ 0.2) ϫ 10 Ϫ4 s Ϫ1 . (B) YYW RNAP. The slow phase of the association reaction (F) has a ka 2 ϭ (1.1 Ϯ 0.3) ϫ 10 Ϫ3 s Ϫ1 ; the slow phase of the dissociation reaction (s), a kd 2 ϭ (6 Ϯ 1) ϫ 10 Ϫ4 s Ϫ1 . Fig. 6. BCL-6 preferentially binds to the wild-type exon 1 in Ly1 cells. Both Ly1 and the control Ly7 cells wer...

BackgroundMicroglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a.ResultsWnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including β-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia.ConclusionThese findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo.

MIR137, transcribed as the microRNA miR-137, is one of the leading candidate schizophrenia susceptibility genes to arise from large genome-wide association studies (GWAS) of the disorder. Recent data suggest that miR-137 modulates the expression of other schizophrenia susceptibility genes. Although bioinformatic resources are available with which to predict genes regulated by individual microRNA, there has been a lack of empirical data on genome-wide gene expression changes following miR-137 manipulation. We have therefore performed a genome-wide assessment of transcriptional changes in a human neural progenitor cell line after miR-137 over-expression and inhibition in order to elucidate molecular pathways by which genetic perturbation of miR-137 could promote susceptibility to schizophrenia. Bioinformatically-predicted miR-137 targets showed a small but highly significant down-regulation following miR-137 over-expression. Genes that were significantly down-regulated in association with miR-137 over-expression were enriched for involvement in neuronal differentiation. Differentially expressed genes that were confirmed by qPCR included others at genome-wide significant risk loci for schizophrenia (MAD1L1 and DPYD) and BDNF. These data point to molecular pathways through which genetic variation at the MIR137 locus could confer risk for schizophrenia.

Increasingly, clinical trials for Alzheimer’s disease (AD) are being conducted earlier in the disease phase and with biomarker confirmation using in vivo amyloid PET imaging or CSF tau and Aβ measures to quantify pathology. However, making such a pre-clinical AD diagnosis is relatively costly and the screening failure rate is likely to be high. Having a blood-based marker that would reduce such costs and accelerate clinical trials through identifying potential participants with likely pre-clinical AD would be a substantial advance. In order to seek such a candidate biomarker, discovery phase proteomic analyses using 2DGE and gel-free LC-MS/MS for high and low molecular weight analytes were conducted on longitudinal plasma samples collected over a 12-year period from non-demented older individuals who exhibited a range of 11C-PiB PET measures of amyloid load. We then sought to extend our discovery findings by investigating whether our candidate biomarkers were also associated with brain amyloid burden in disease, in an independent cohort. Seven plasma proteins, including A2M, Apo-A1, and multiple complement proteins, were identified as pre-clinical biomarkers of amyloid burden and were consistent across three time points (p < 0.05). Five of these proteins also correlated with brain amyloid measures at different stages of the disease (q < 0.1). Here we show that it is possible to detect a plasma based biomarker signature indicative of AD pathology at a stage long before the onset of clinical disease manifestation. As in previous studies, acute phase reactants and inflammatory markers dominate this signature.